Drug resistance, along with normal tissue toxicity, remains a major obstacle for the successful treatment of cancer. Targeting these resistance mechanisms will result in increased tumor cell death, decreased tumor size, and may lead to overall patient survival. The purpose of this study is to understand how hyperthermia (HT) alters cellular resistance mechanisms and to exploit these abilities to increase drug delivery to the tumor site. The work outlined in this application will focus on targeting inefficiencies in drug transport and inherent cellular resistance mechanisms. Specifically, we propose to combine the many therapeutic benefits of HT with copper chelators and cisplatin-encapsulated thermosensitive liposomes (TSL) to target and treat muscle invasive disease in bladder cancer patients. Our group has recently developed a TSL that encapsulates cisplatin. Using this drug formulation in combination with HT is a modality that will overcome inefficiencies in drug transport, and reduce the undesirable toxicity to other tissues. HT is known to interact synergistically with multiple chemotherapeutic drugs, including cisplatin, but the molecular mechanisms underlying this interaction remain unclear. Copper transporter protein 1 (Ctr1) is a major regulator of cellular cisplatin uptake. Our preliminary data show that HT increases cisplatin uptake and platinum-DNA adduct formation in wild type but not in Ctr1-/- cells indicating the involvement of Ctr1 in this synergism. One goal of our study is to use copper chelators to create an environment of copper deficiency in order to increase Ctr1 expression levels on the cell surface. Increased Ctr1 membrane expression in the presence of cisplatin will lead to increased drug uptake, accumulation, and cytotoxicity. Enhancing cisplatin uptake and efficacy by increasing Ctr1 expression via copper chelation has yet to be studied and could prove to be an effective treatment modality with clinical applicability and potential. We hypothesize that utilizing the combination of a TSL-based system paired with Ctr1 manipulation will increase cisplatin delivery and uptake in tumor cells and will prove to be an effective anti-cancer strategy. We will address our hypothesis with two Specific Aims. In vitro, we plan to elucidate the role Ctr1 plays in the synergistic interaction between cisplatin and HT and use this knowledge to increase drug efficacy. In vivo, we will examine the pharmacokinetics and anti-tumor effect of the cisplatin-TSL and HT combination and apply the information derived from the Ctr1 studies to improve this combinational treatment to increase the overall anti- cancer effect. Since cisplatin and copper chelators are currently both FDA approved drugs, our proposed treatment regime should have a relatively straightforward transition into the clinic. PUBLIC HEALTH RELEVANCE: Muscle invasive disease (MID) accounts for approximately 33% of the newly diagnosed bladder cancer patients, and 10-15% of patients with superficial disease will eventually progress to MID. Approximately 50% of MID patients develop metastases which in turn significantly decreases survival rates. Current standard of care for MID involves neo-adjuvant chemotherapy with a cisplatin containing regimen followed by radical cystectomy. Using a novel combination of regional hyperthermia (HT), cisplatin-thermosensitive liposome (TSL), and copper chelation, our goal is to develop a treatment strategy for MID that will both positively impact patient survival and improve patient quality of life by allowing for bladder preservation. HT is a well-tolerated, useful adjuvant treatment that increases drug delivery and drug targeting, particularly in the case of liposomal therapeutics by acting as a focal drug release trigger for TSL. Cisplatin synergistically interacts with HT resulting in increased cytotoxicity. Our proposed work seeks to develop and optimize a unique combination of treatments that will overcome three barriers of drug efficacy: drug delivery, cellular drug resistance, and normal tissue toxicity. We will use the novel cisplatin-TSL combined with HT to increase tumor drug delivery and decrease normal tissue toxicity and copper chelation and HT to manipulate the cisplatin cellular transporter Ctr1 to increase cellular cisplatin accumulation and cytotoxicity.